Tyrosine sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2), that catalyze the transfer of sulfate from PAPS to lumenally oriented tyrosine residues in secreted and transmembrane proteins. A small number of proteins are known to be tyrosine sulfated in man, including certain adhesion molecules, coagulation factors, serpins, extracellular matrix proteins, and hormones and some of these require sulfation for optimal function. Recently, several G-protein-coupled receptors (GPCRs), including the chemokine receptors CCR2B, CXCR4, CCR5, and CX3CR1, the C5a receptor, and the TSH receptor were shown to be tyrosine sulfated on their N-terminal domains and to require sulfation for optimal binding of their cognate ligands. However, all current data on GPCR sulfation are based on human receptors expressed in cell lines. Thus, the importance of GPCR sulfation in ligand binding in native cells and its relevance in vivo is uncertain. The N-terminal domains of all known chemokine receptors (CCRs) contain tyrosine residues in the context of several flanking acidic amino acids, features shared by all known tyrosine sulfated proteins. This suggests that all CCRs may be tyrosine sulfated and require sulfation for chemokine binding. The goal of this application is to systematically examine whether mouse CCRs are tyrosine sulfated and to explore the role of tyrosine sulfation in CCR function in vitro and in vivo. The following specific aims are proposed. Aim 1 -Determine if the known CCRs are tyrosine sulfated in the mouse. Aim 2 - Determine if CCR sulfation is required for optimal function in vitro by assessing chemokine-induced responses of cells derived fromTpst1-/- and Tpst2-/- mice. Aim 3 - Determine the biological relevance of deficient sulfation of CCRs in vivo by examining biological responses to inflammatory challenges that normally require a particular CCR are abnormal in Tpst1-/- and Tpst2-/- mice. Chemokines and their receptors play crucial roles in a variety of pathophysiological processes, such as bacterial and viral infection (i.e. HIV/AIDS), allergy, atherosclerosis, malignancy, and various autoimmune and chronic inflammatory diseases. These studies will provide new insights into the importance of tyrosine sulfation in innate and adaptive host responses during a wide variety of pathophysiological processes.